Abundance of identified bacterial species belonging to their respective family isolated from the midgut of adult sugar fed female, blood fed female, male and larvae of <i>Aedes albopictus</i>.

<p>Abundance of identified bacterial species belonging to their respective family isolated from the midgut of adult sugar fed female, blood fed female, male and larvae of <i>Aedes albopictus</i>.</p

Diversity of Cultivable Midgut Microbiota at Different Stages of the Asian Tiger Mosquito, <i>Aedes albopictus</i> from Tezpur, India

<div><p><i>Aedes aegypti</i> and <i>Ae</i>. <i>albopictus</i> are among the most important vectors of arboviral diseases, worldwide. Recent studies indicate that diverse midgut microbiota of mosquitoes significantly affect development, digestion, metabolism, and immunity of their hosts. Midgut microbiota has also been suggested to modulate the competency of mosquitoes to transmit arboviruses, malaria parasites etc. Interestingly, the midgut microbial flora is dynamic and the diversity changes with the development of vectors, in addition to other factors such as species, sex, life-stage, feeding behavior and geographical origin. The aim of the present study was to investigate the midgut bacterial diversity among larva, adult male, sugar fed female and blood fed female <i>Ae</i>. <i>albopictus</i> collected from Tezpur, Northeastern India. Based on colony morphological characteristics, we selected 113 cultivable bacterial isolates for 16S rRNA gene sequence based molecular identification. Of the 113 isolates, we could identify 35 bacterial species belonging to 18 distinct genera under four major phyla, namely Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Phyla Proteobacteria and Firmicutes accounted for majority (80%) of the species, while phylum Actinobacteria constituted 17% of the species. Bacteroidetes was the least represented phylum, characterized by a single species- <i>Chryseobacterium rhizoplanae</i>, isolated from blood fed individuals. Dissection of midgut microbiota diversity at different developmental stages of <i>Ae</i>. <i>albopictus</i> will be helpful in better understanding mosquito-borne diseases, and for designing effective strategies to manage mosquito-borne diseases.</p></div

Occurrence of identified bacterial species from the midgut of A. sugar fed female, B. blood fed female, C. male and D. larvae <i>Ae</i>. <i>albopictus</i>.

<p>Occurrence of identified bacterial species from the midgut of A. sugar fed female, B. blood fed female, C. male and D. larvae <i>Ae</i>. <i>albopictus</i>.</p

Diversity indices, total taxa, and Good’s coverage of midgut bacterial isolates of <i>Ae</i>. <i>albopictus</i> from different life stages.

<p>Diversity indices, total taxa, and Good’s coverage of midgut bacterial isolates of <i>Ae</i>. <i>albopictus</i> from different life stages.</p

Abundance of identified bacterial species belonging to their respective phylum isolated from the midgut of adult sugar fed female, blood fed female, male, and larvae of <i>Aedes albopictus</i>.

<p>Abundance of identified bacterial species belonging to their respective phylum isolated from the midgut of adult sugar fed female, blood fed female, male, and larvae of <i>Aedes albopictus</i>.</p

Phylogenetic analysis of bacterial isolates from midgut of <i>Ae</i>. <i>albopictus</i>.

<p>Phylogenetic tree based on partial 16S rRNA gene sequences, reconstructed through neighbor joining algorithm using Kimura 2 distance parameter method. The percentage bootstrap values obtained with 1000 replications are denoted at branch node.</p

IFN-α and RBV each inhibited the internal ribosome entry site (IRES) mediated translation of green fluorescence protein (GFP).

<p>Huh-7 cells were infected with T7-expressing adenovirus. After 2 hrs, HCV IRES-GFP plasmid was transfected and then treated with indicated concentration of IFN-α and RBV. (<b>A</b>) HCV IRES mediated GFP expression was monitored under fluorescent microscopy. (<b>B</b>) Inhibition of GFP expression was further confirmed by Western blot analysis in both IRES and non-IRES mechanisms. β-actin is used as loading controls.</p

Different combinations of IFN-α, RBV, and IFN-λ inhibits HCV IRES Rluc mediated translation.

<p>Huh-7 cells were infected with T7-expressing adenovirus. After 2 hrs, HCV IRES-RLuc plasmid was transfected and then treated with indicated concentration of IFN-α, IFN-λ and RBV. The concentration dependent inhibition of <i>Renilla</i> luciferase activity by (<b>A</b>) IFN-α, RBV, and IFN-λ single treatment;(<b>B</b>) Combination of IFN-α + IFN-λ; (<b>C</b>) Combination of IFN-α + RBV and (<b>D</b>) Combination of IFN-λ + RBV.</p

IFN-α and RBV synergy antiviral mechanism involves the activation of PKR, eIF2α and inhibition of cellular IMPDH.

<p>(<b>A</b>) IFN-α and RBV each induced phosphorylation of PKR and eIF2α. (<b>B</b>) Flow cytometric analysis showing RBV show a dose dependent inhibition of HCV IRES-GFP translation. (<b>C</b>) Inhibition of IMPDH and PKR levels by siRNA prevented RBV antiviral action against HCV IRES-GFP translation determined by flow cytometric analysis. (<b>D</b>) Dose dependent prevention of RBV action due to increasing concentration of guanosine was determined by flow cytometric analysis. (<b>E</b>) IFN-α inhibits HCV IRES-GFP translation. (<b>F</b>) Inhibition of PKR by siRNA prevented IFN-α mediated inhibition of HCV IRES-GFP translation.</p

Antiviral effect of IFN-α and RBV combination treatment using a sub-genomic replicon cell line (S3-GFP) and HCV infected Huh-7.5 cells.

<p>(<b>A</b>) S3-GFP cells were treated with IFN-α and RBV for 72 hours. GFP expression was examined under a fluorescence microscope. (<b>B</b>) GFP positive cells were quantified by flow cytometric analysis. (<b>C</b>) Infected Huh-7.5 cells were treated with IFN-α alone, RBV alone and combination for 72 hours. <i>Renilla</i> Luciferase activity of infected cells was measured and normalized with 1µg of cellular protein. (<b>D</b>) Expression of HCV core protein was measured by immunostaining and (<b>E</b>) core positive cells in five different high power fields (hpf) at 40X magnification were counted under a light microscope. Quantitative assessment of the number of HCV positive cells with mean and standard deviation of the combination treatment are compared.</p